Metabolic diseases, including type 2 diabetes and non-alcoholic fatty liver disease, arise from disordered energy utilization and storage, and are therefore directly linked to the physiology of adipose tissue. Many adult humans possess metabolically active beige adipose tissue, and its mass shows an inverse correlation with adiposity and a direct correlation with metabolic health. How this tissue arises, responds to environmental cues, and its potential for diagnostic and therapeutic use are exciting questions currently under investigation. However, adequate models to study this tissue from humans are lacking. This proposal is based on an exciting recent finding from our laboratory that enables the isolation and expansion of human primary pre-adipocytes that give rise to white and beige adipocytes. We discovered that human beige pre-adipocytes are localized within the adipose tissue vasculature, and proliferate only under conditions that promote capillary network expansion in vitro. We now seek to leverage these findings to understand the cell and molecular basis for the formation of these cells, and the mechanisms underlying their beneficial metabolic effects. Specific Aim 1: Using new deep sequencing and bioinformatics approaches that enable cell annotation using very low RNA input we will determine whether white and beige adipocytes emerge from the same or distinct pre-adipocyte progenitors. Specific Aim 2: Our preliminary studies indicate that implantation of human beige adipocytes into NOD-scid IL2r?null (NSG) mice improves glucose disposal, suggesting that cells per-se can confer a metabolic benefit. We will determine whether implanted human beige adipocytes improve systemic glucose metabolism by acting as an energy sink for glucose and lipid utilization, or by secreting factors that enhance glucose tolerance and insulin sensitivity in other tissues. Specific Aim 3: Our new findings from gene expression analysis of human beige adipocytes has revealed induction of mRNAs for potent neuroendocrine factors, including the pro-protein convertase PCSK1 which is genetically associated with obesity in numerous human populations. Thus, the beneficial effect of human beige adipocytes may be due to a neuroendocrine function. Mass spectroscopy has confirmed the secretion of fragments of PCSK1, and identified novel secreted peptides. Proteomic analysis proposed here will further define the secreted proteome from these cells, and provide information necessary for analyzing its impact on systemic glucose tolerance. The work proposed will answer pivotal questions regarding the origin, characteristics and functional properties of human beige adipocytes and their secreted products, and provide the foundation for clinical studies leveraging these findings for diagnostic and therapeutic applications in human metabolic disease.